Introduced in Mrs. Frisby and the Rats of NIMH, Jonathan was one of eight mice experimented on at NIMH, where they received the same injections as the future Rats of NIMH. This resulted in them gaining enhanced intelligence, and a greater lifespan than normal mice. Jonathan and his comrades were kept in small cages within a large cabinet, from which they were unable to make their escape. However, upon overhearing the rats' plans to escape through the air vents of the NIMH facility, Jonathan boldly asked them if they would free the mice and allow them to make their escape as well.

The rats' leader, Nicodemus, consented to this, on the condition that the mice go their own way at the first opportunity. However, during the escape attempt, disaster struck, with the air conditioning system blowing most of Jonathan's fellows away. The rats were only able to rescue Jonathan and Mr. Ages, and dissuaded them from attempting to find the missing mice themselves. The rat Justin attempted to locate the lost ones while Jonathan and Mr. Ages helped the rats open a grating to enable their escape.

With the failure to find the other mice, the rats kindly invited Jonathan and Mr. Ages to remain with them, which they did for a number of years. Their path together would take them to the Boniface estate and later the discovery of the Toy Tinker and his truck. However, after the rats established a home for themselves on the Fitzgibbons farm, the two mice moved out on their own. For a time Jonathan lived in Mr. Ages' house with him, but then he met the future Mrs. Frisby at a stream.

Jonathan married, and he and Mrs. Frisby had four children: Martin, Teresa, Timothy, and Cynthia. When Timothy was very young he was bitten by a spider, and Jonathan and Mrs. Frisby took him to see Mr. Ages, who was able to give him an antidote. Jonathan also taught his wife and children to read as he had learned, and told Mrs. Frisby about the many things he had learned about. However, he dreaded the fact that his increased lifespan would see him remain young while his wife grew old, and agonized over how to tell her.

Jonathan also maintained his association with the rats, taking pains to hide this from his family. He would also take part in their missions to slip sleeping powder into the food of Dragon, the Fitzgibbons' fierce cat. Unfortunately, during one such mission Jonathan perished, leaving his family bereaved. Mrs. Frisby would not learn the truth about his fate until a year later, when she approached the rats for help and learned the full story about his relationship with the rats.